The hair follicle undergoes a life-long transformation from a resting phase (telogen) to a growth phase (anagen) with rapid proliferation of follicular keratinocytes and elongation and thickening of the hair shaft. Anagen phase is followed by regression phase (catagen) leading to involution of the hair follicle (telogen) which continues until a new hair shaft is generated in the existing follicle during the subsequent anagen phase. Hardy et al. (1992) Trends in Genetics 8:55-61. These cyclic changes involve rapid remodeling of both epithelial and dermal components of the hair follicle.
The invention is based, in part, on the discovery that increased perifollicular vascularization promotes hair growth and that expression of VEGF by follicular keratinocytes leads to increased perifollicular vascularization. In addition, it was found that increasing the level of VEGF expression resulted in accelerated hair regrowth and increased hair follicle size, which leads to hair thickening. It was also found that by inhibiting the levels of VEGF, hair growth and hair thickening can be reduced.
Accordingly, in one aspect, the invention features a method of modulating hair growth and/or hair thickness. The method includes modulating VEGF protein, e.g., modulating VEGF gene expression and/or modulating VEGF protein production and/or activity, to thereby modulate hair growth and/or thickness.
In another aspect, the invention features a method of promoting hair growth in a subject. The method includes increasing the VEGF activity, e.g., increasing the level of VEGF protein, e.g., increasing the levels of VEGF gene expression and/or increasing VEGF protein production and/or activity, to thereby promote hair growth.
In a preferred embodiment, VEGF activity is increased by administering an agent which increases the level of VEGF protein. An agent which increases the level of VEGF protein can be one or more of: a VEGF polypeptide or a functional fragment or analog thereof; a nucleotide sequence encoding a VEGF polypeptide or functional fragment or analog thereof; an agent which increases VEGF nucleic acid expression, e.g., a transition metal ion, or a small molecule which binds to the promoter region of VEGF.
In a preferred embodiment, VEGF is increased by administering, e.g., introducing, a nucleotide sequence encoding a VEGF polypeptide or functional fragment or analog thereof, into a particular cell, e.g., a keratinocyte, e.g., a follicular keratinocyte, in the subject. The nucleotide sequence can be a genomic sequence or a cDNA sequence. The nucleotide sequence can include: a VEGF coding region; a promoter sequence, e.g., a promoter sequence from a VEGF gene or from another gene; an enhancer sequence, e.g., 5xe2x80x2 untranslated region (UTR), e.g., a 5xe2x80x2 UTR from a VEGF gene or from another gene, a 3xe2x80x2 UTR, e.g., a 3xe2x80x2 UTR from a VEGF gene or from another gene; a polyadenylation site; an insulator sequence.
In another preferred embodiment, the agent is a compound, e.g., a small molecule, which increases VEGF expression. For example, the molecule can be a transition metal ion, e.g., manganese, cobalt, nickel, or combinations thereof. In another preferred embodiment, the agent is a polypeptide other than VEGF which increases VEGF expression. For example, the agent can be a cytokine, e.g., interleukin-1, or a growth factors, e.g., transforming growth factor-xcex1, epidermal growth factor.
In another preferred embodiment, the level of VEGF protein is increased by increasing the level of expression of an endogenous VEGF coding sequence, e.g., by increasing transcription of the VEGF coding sequence. In a preferred embodiment, transcription of the VEGF gene is increased by: altering the regulatory sequences of the endogenous VEGF gene, e.g., by the addition of a positive regulatory element (such as an enhancer or a DNA-binding site for a transcriptional activator); the deletion of a negative regulatory element (such as a DNA-binding site for a transcriptional repressor) and/or replacement of the endogenous regulatory sequence, or elements therein, with that of another gene, thereby allowing the coding region of the VEGF gene to be transcribed more efficiently.
In another preferred embodiment, the method can include introducing a cell, e.g., a cell which expresses and preferably secretes a VEGF protein, into a subject. In a preferred embodiment, the cell has been genetically modified to express a VEGF protein, a fragment or an analog thereof, or a protein other than VEGF which causes an increase in the levels of VEGF. The cell can be an autologous, allogeneic, or xenogeneic cell, but is preferably autologous. In a preferred embodiment, the cell is encapsulated, e.g., in a gel or biocompatible mesh, which is introduced into the subject. The cell can be any cell type, e.g., a fibroblast, a keratinocyte, an epithelial cell, an endothelial cell. Preferably the cell is a keratinocyte, e.g., a follicular keratinocyte. The cell can be introduced into a subject to increase the level of VEGF protein.
In a preferred embodiment, the agent which increases the level of VEGF protein is administered, e.g., by topically administering the agent; systemically administering the agent; orally administering the agent; or injecting the agent, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation. Preferably, the agent is included in a composition for topical use, e.g., the composition is a gel, cream, or liquid. In a preferred embodiment, the agent is administered: by continuous administration, e.g., the agent is administered with sufficient frequency such that the affect on the VEGF protein level is maintained for a selected period, e.g., 10, 20, 30, 50, 90, 180, 365 days or more. In another preferred embodiment, administration of the agent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20 or more times.
In a preferred embodiment, hair growth is promoted on: the subject""s scalp; the subject""s face, e.g., beard and/or mustache facial hair growth is promoted.
In a preferred embodiment, the subject has an insufficient amount of hair or an insufficient rate of hair growth. In a preferred embodiment, the subject suffers from genetic pattern baldness; suffers from a hormonal disorder which decreases hair growth; has received a treatment, e.g., radiation, or chemotherapy, or a drug which inhibits hair growth; or has had a surgical procedure, e.g., skin graft, which is in need of hair growth.
In another aspect, the invention features a method of enhancing hair thickness. The method includes increasing the level of VEGF protein, e.g., increasing the levels of VEGF gene expression and/or increasing VEGF protein production and/or activity, to thereby promote thickening of the hair.
In a preferred embodiment, VEGF activity is increased by administering an agent which increases the level of VEGF protein. An agent which increases the level of VEGF protein can be one or more of: a VEGF polypeptide or a functional fragment or analog thereof; a nucleotide sequence encoding a VEGF polypeptide or functional fragment or analog thereof; an agent which increases VEGF nucleic acid expression, e.g., a transition metal ion, or a small molecule which binds to the promoter region of VEGF.
In a preferred embodiment, VEGF is increased by administering, e.g., introducing, a nucleotide sequence encoding a VEGF polypeptide or functional fragment or analog thereof, into a particular cell, e.g., a keratinocyte, e.g., a follicular keratinocyte, in the subject. The nucleotide sequence can be a genomic sequence or a cDNA sequence. The nucleotide sequence can include: a VEGF coding region; a promoter sequence, e.g., a promoter sequence from a VEGF gene or from another gene; an enhancer sequence, e.g., 5xe2x80x2 untranslated region (UTR), e.g., a 5xe2x80x2 UTR from a VEGF gene or from another gene, a 3xe2x80x2 UTR, e.g., a 3xe2x80x2 UTR from a VEGF gene or from another gene; a polyadenylation site; an insulator sequence.
In preferred embodiment, the agent is a compound, e.g., a small molecule, which increases VEGF expression. For example, the molecule can be a transition metal ion, e.g., manganese, cobalt, nickel, or combinations thereof, or an inducer of reactive oxygen species. In another preferred embodiment, the agent is a polypeptide other than VEGF which increases VEGF expression. For example, the agent can be a cytokine, e.g., interleukin-1, or a growth factors, e.g., transforming growth factor-xcex1, epidermal growth factor.
In another preferred embodiment, the level of VEGF protein is increased by increasing the level of expression of an endogenous VEGF gene, e.g., by increasing transcription of the VEGF gene. In a preferred embodiment, transcription of the VEGF gene is increased by: altering the regulatory sequences of the endogenous VEGF gene, e.g., by the addition of a positive regulatory element (such as an enhancer or a DNA-binding site for a transcriptional activator); the deletion of a negative regulatory element (such as a DNA-binding site for a transcriptional repressor) and/or replacement of the endogenous regulatory sequence, or elements therein, with that of another gene, thereby allowing the coding region of the VEGF gene to be transcribed more efficiently.
In another preferred embodiment, the method can include introducing a cell, e.g., a cell which expresses and preferably secretes a VEGF protein, into a subject. In a preferred embodiment, the cell has been genetically modified to express a VEGF protein, or a fragment or an analog thereof. The cell can be an autologous, allogeneic, or xenogeneic cell, but is preferably autologous. The cell can be any cell type, e.g., a fibroblast, a keratinocyte, an epithelial cell, an endothelial cell. Preferably the cell is a keratinocyte, e.g., a follicular keratinocyte. The cell can be encapsulated, e.g., in a gel or biocompatible mesh. The cell can be introduced into a subject to increase the level of VEGF protein.
In a preferred embodiment, the agent which increases the level of VEGF protein is administered, e.g., by topically administering the agent; systemically administering the agent; orally administering the agent; or injecting the agent, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation. Preferably, the agent is included in a composition for topical use, e.g., the composition is a gel, cream, or liquid. In a preferred embodiment, the agent is administered: by continuous administration, e.g., the agent is administered with sufficient frequency such that the affect on the VEGF protein level is maintained for a selected period, e.g., 10, 20, 30, 50, 90, 180, 365 days or more. In another preferred embodiment, administration of the agent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20 or more times.
In a preferred embodiment, hair thickness is promoted on: the subject""s scalp; the subject""s face, e.g., beard and/or mustache.
In a preferred embodiment, the subject has: fine or limp hair; an insufficient amount of hair; an insufficient rate of hair growth.
In another aspect, the invention features a method of inhibiting hair growth in a subject. The method includes inhibiting the level of VEGF activity, e.g., inhibiting the level of VEGF protein, decreasing the levels of VEGF gene expression and/or decreasing VEGF protein production and/or activity, in the subject.
In a preferred embodiment, VEGF is inhibited by administering an agent which inhibits VEGF. An agent which inhibits VEGF can be one or more of: a VEGF nucleic acid molecule which can bind to a cellular VEGF nucleic acid sequence, e.g., mRNA, and inhibit expression of the protein, e.g., an antisense molecule or VEGF ribozyme; an antibody that specifically binds to VEGF protein, e.g., an antibody that disrupts VEGF""s ability to bind to its natural cellular target; an agent which decreases VEGF gene expression, e.g., a small molecule which binds the promoter of VEGF.
In another preferred embodiment, VEGF activity is inhibited by decreasing the level of expression of an endogenous VEGF gene, e.g., by decreasing transcription of the VEGF gene. In a preferred embodiment, transcription of the VEGF gene can be decreased by: altering the regulatory sequences of the endogenous VEGF gene, e.g., by the addition of a negative regulatory sequence (such as a DNA-binding site for a transcriptional repressor).
In another preferred embodiment, the agent is a compound, e.g., small molecule, which is known to inhibit VEGF, e.g., a transition metal.
In a preferred embodiment, the agent which inhibits VEGF expression is administered, e.g., by topically administering the agent; systemically administering the agent; orally administering the agent; or injecting the agent, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation. Preferably, the agent is included in a composition for topical use, e.g., the composition is a gel, cream, or liquid. In a preferred embodiment, the agent is administered: by continuous administration, e.g., the agent is administered with sufficient frequency such that the affect on the VEGF protein level is maintained for a selected period, e.g., 10, 20, 30, 50, 90, 180, 365 days or more. In another preferred embodiment, administration of the agent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20 or more times.
In a preferred embodiment, hair growth is inhibited on: the subject""s scalp; the subject""s face, e.g., beard and/or mustache facial hair growth or eyebrow growth is inhibited; the subject""s body hair growth is inhibited, e.g., hair growth is inhibited on the subject""s back, legs, chest, armpits.
In a preferred embodiment, the method further includes the removal of hair, e.g., by plucking, shaving or application of a depilatory. In a preferred embodiment, hair is removed sequentially or simultaneously with the inhibition VEGF expression. Preferably, hair is removed prior to the inhibition of VEGF expression.
In another aspect, the invention features a method of reducing hair thickness. The method includes inhibiting the level of VEGF protein, e.g., decreasing the levels of VEGF gene expression and/or decreasing VEGF protein production and/or activity in the subject.
In a preferred embodiment, VEGF is inhibited by administering an agent which inhibits VEGF. An agent which inhibits VEGF can be one or more of: a VEGF nucleic acid molecule which can bind to a cellular VEGF nucleic acid sequence, e.g., mRNA, and inhibit expression of the protein, e.g., an antisense molecule or VEGF ribozyme; an antibody that specifically binds to VEGF protein, e.g., an antibody that disrupts VEGF""s ability to bind to its natural cellular target; an agent which decreases VEGF gene expression, e.g., a small molecule which binds the promoter of VEGF.
In another preferred embodiment, VEGF is inhibited by decreasing the level of expression of an endogenous VEGF gene, e.g., by decreasing transcription of the VEGF gene. In a preferred embodiment, transcription of the VEGF gene can be decreased by: altering the regulatory sequences of the endogenous VEGF gene, e.g., by the addition of a negative regulatory sequence (such as a DNA-binding site for a transcriptional repressor).
In another preferred embodiment, the agent is a compound, e.g., small molecule, which is known to inhibit VEGF.
In a preferred embodiment, the agent which inhibits VEGF expression is administered, e.g., by topically administering the agent; systemically administering the agent; orally administering the agent; or injecting the agent, preferably dermally or subcutaneously. In preferred embodiments, the compound is administered using a suitable delivery vehicle, for example, a surfactant or an agent which increases permeability in the skin, e.g., an SDS or DMSO containing formulation. Preferably, the agent is included in a composition for topical use, e.g., the composition is a gel, cream, or liquid. In a preferred embodiment, the agent is administered: by continuous administration, e.g., the agent is administered with sufficient frequency such that the affect on the VEGF protein level is maintained for a selected period, e.g., 10, 20, 30, 50, 90, 180, 365 days or more. In another preferred embodiment, administration of the agent is repeated, e.g., is repeated at least 1, 2, 3, 5, 10, 20 or more times.
In a preferred embodiment, hair thickness is reduced on: the subject""s scalp; the subject""s face, e.g., beard and/or mustache or eyebrows; the subject""s body, e.g., on the subject""s back, legs, chest, armpits.
In a preferred embodiment, the method further includes the removal of hair, e.g., by plucking, shaving or application of a depilatory. In a preferred embodiment, hair is removed sequentially or simultaneously with the inhibition VEGF expression. Preferably, hair is removed prior to the inhibition of VEGF expression.
In another aspect, the invention features a method of evaluating the status of hair growth/hair loss in a subject. The method includes evaluating, e.g., detecting, the presence or absence of a genetic lesion in a VEGF gene, or evaluating, e.g., detecting, misexpression of the VEGF gene.
In one embodiment, the method includes evaluating whether a subject is at risk for hair loss. The method includes evaluating, e.g., detecting, a genetic lesion in a VEGF gene, or evaluating, e.g., detecting, underexpression of the VEGF gene, to thereby determine if a subject is at risk for hair loss.
In preferred embodiment, the method includes evaluating in a sample of cells from the subject for the presence or absence of a genetic lesion, e.g., a mutation in the gene encoding a VEGF protein. The presence of a genetic lesion is indicative of a risk of hair loss in a subject. The cell sample can be of any cell type, e.g., a fibroblast, a keratinocyte, an epithelial cell, an endothelial cell, a glial cell, a neural cell, a lymphocyte, a bone marrow cell, and a muscle cell.
In another preferred embodiment, the method includes evaluating in a sample of cells, e.g., a sample of keratinocytes, e.g., follicular keratinocytes, of a subject, for the expression levels of the VEGF to determine underexpression. Underexpression of VEGF can be indicative of a risk of hair loss.
In a preferred embodiment, the genetic lesions is evaluated by contacting the sample with a nucleic acid probe capable of hybridizing to VEGF mRNA, e.g., a labeled probe. In another preferred embodiment, expression of VEGF is evaluated with an antibody capable of binding to VEGF protein, e.g., a labeled antibody.
In another embodiment, the method includes evaluating hair growth in a subject. The method includes evaluating, e.g., detecting, absence or presence of a genetic lesion in a VEGF gene, or evaluating, e.g., detecting, overexpression of the VEGF gene, to thereby evaluate whether hair growth is likely in a subject.
In a preferred embodiment, the method includes evaluating in a sample of cells from the subject for the presence or absence of a genetic lesion, e.g., a mutation in the gene encoding a VEGF protein. The absence of a genetic lesion can be indicative of a potential for hair growth. The cell sample can be of any cell type, e.g., a fibroblast, a keratinocyte, an epithelial cell, an endothelial cell, a glial cell, a neural cell, a lymphocyte, a bone marrow cell, and a muscle cell.
In another preferred embodiment, the method includes evaluating in a sample of cells, e.g., a sample of keratinocytes, e.g., follicular keratinocytes, of a subject, for the expression levels of VEGF to determine expression. Normal levels of VEGF expression or overexpression of VEGF can be indicative of a potential for hair growth.
In a preferred embodiment, the genetic lesions is evaluated by contacting the sample with a nucleic acid probe capable of hybridizing to VEGF mRNA, e.g., a labeled probe. In another preferred embodiment, expression of VEGF is evaluated with an antibody capable of binding to VEGF protein, e.g., a labeled antibody.
In another aspect, the invention features a method of evaluating the ability of a keratinocyte to induce growth or hair loss in a subject. The method includes evaluating, e.g., detecting, the presence or absence of a genetic lesion in a VEGF gene, or evaluating, e.g., detecting, misexpression of the VEGF gene.
In a preferred embodiment, the keratinocyte is evaluated in vitro.
In one embodiment, the method includes evaluating the ability of a keratinocyte to induce hair loss. The method includes evaluating, e.g., detecting, a genetic lesion in a VEGF gene, or evaluating, e.g., detecting, underexpression of the VEGF gene.
In preferred embodiment, the method includes evaluating a sample of keratinocytes, e.g., follicular kertinocytes, for the presence or absence of a genetic lesion, e.g., a mutation in the gene encoding a VEGF protein. The presence of a genetic lesion is indicative of a risk of hair loss in a subject.
In another preferred embodiment, the method includes evaluating a sample of keratinocytes, e.g., follicular keratinocytes, of a subject, for the expression levels of the VEGF to determine underexpression. Underexpression of VEGF is indicative of a risk of hair loss.
In a preferred embodiment, the genetic lesions is evaluated by contacting the sample with a nucleic acid probe capable of hybridizing to VEGF mRNA, e.g., a labeled probe. In another preferred embodiment, expression of VEGF is evaluated with an antibody capable of binding to VEGF protein, e.g., a labeled antibody.
In another embodiment, the method includes evaluating the ability of a kertinocyte to induce hair growth. The method includes evaluating, e.g., detecting, absence or presence of a genetic lesion in a VEGF gene, or evaluating, e.g., detecting, overexpression of the VEGF gene.
In a preferred embodiment, the method includes evaluating in a sample of keratinocytes, e.g., follicular keratinocytes, from the subject for the presence or absence of a genetic lesion, e.g., a mutation in the gene encoding a VEGF protein. The absence of a genetic lesion is indicative of a potential for hair growth.
In another preferred embodiment, the method includes evaluating in a sample of keratinocytes, e.g., follicular keratinocytes, of a subject, for the expression levels of VEGF to determine expression. Normal levels of VEGF expression or overexpression of VEGF is indicative of a potential for hair growth.
In a preferred embodiment, the genetic lesions is evaluated by contacting the sample with a nucleic acid probe capable of hybridizing to VEGF mRNA, e.g., a labeled probe. In another preferred embodiment, expression of VEGF is evaluated with an antibody capable of binding to VEGF protein, e.g., a labeled antibody.
In another aspect, the invention features, a method of evaluating a candidate compound. The method is useful for identifying a compound, e.g., a VEGF polypeptide, or a fragment or analog thereof, which can be used to modulate hair growth and/or thickness. The method can evaluate the ability of the compound to increase VEGF activity, e.g., by increasing the expression of the VEGF gene or the activity of the VEGF protein. The method includes: providing a cell, a tissue, or a subject, treating the cell or the tissue, or the subject with a candidate compound; and determining the level of VEGF RNA, VEGF DNA or VEGF protein. The method can further include evaluating a control cell, tissue or subject, e.g., an identical cell which, e.g., is not treated with the candidate compound. An increase in the amount of VEGF activity in the cell tissue or subject treated with the compound in comparison to the control is indicative of a useful compound, e.g., a compound useful for promoting hair growth and/or thickness.
In a preferred embodiment the compound is a fragment or an analog of VEGF.
In a preferred embodiment, the cell is a keratinocyte, e.g., a follicular keratinocyte.
The invention also features methods for identifying a compound which interacts with a VEGF protein. In a preferred embodiment, the method can include the steps of contacting the VEGF protein with the compound under conditions which allow binding of the compound to the VEGF protein to form a complex, and detecting the formation of a complex of the VEGF protein and the compound in which the ability of the compound to bind to the VEGF protein is indicated by the presence of the compound in the complex. Using such methods, compounds can be identified which modulate, e.g., promote or inhibit, hair growth and/or hair thickness. Methods for identifying a compound or agent can be performed, for example, using a cell free assay. For example, VEGF can be immobilized to a suitable substrate, e.g., glutathione sepharose beads or glutathione derivatized microtitre plates, using a fusion protein which allows for VEGF to bind to the substrate, e.g., a glutathoine-S-transferase/VEGF fusion protein.
In a preferred embodiment the compound is a fragment or an analog of VEGF.
In a preferred embodiment, the compound is a transition metal ion.
In a preferred embodiment, the method further includes providing a cell, a tissue, or a subject, treating the cell or the tissue, or the subject with the identified compound; and determining the level of VEGF RNA, VEGF DNA or VEGF protein. In a preferred embodiment, the cell is a keratinocyte, e.g., a follicular keratinocyte.
In another embodiment, a compound which interacts with a VEGF protein can be identified using a cell-based assay. These methods can include identifying a compound based on its ability to promote, a biological activity of VEGF. In a preferred embodiment, the compound modulates a biological activity of VEGF, e.g., the compound modulates perifollicular vascularization. In a preferred embodiment, the compound is a fragment or an analog of VEGF.
In another aspect, the invention features, a method for identifying compounds which increase VEGF nucleic acid expression. In a preferred embodiment, nucleic acid expression can be evaluated using a nucleic acid probe, e.g., a labeled probe, capable of hybridizing to a VEGF nucleic acid molecule, e.g., VEGF mRNA. In another preferred embodiment, VEGF nucleic acid expression, e.g., DNA expression, can be evaluated by contacting a compound with a VEGF nucleic acid molecule, e.g., a regulatory sequence of a VEGF nucleic acid molecule, and evaluating VEGF transcription, in vitro or in vivo. VEGF transcription can be evaluated, for example, by detecting the production of VEGF protein, e.g., using an antibody, e.g., a labeled antibody, or by determining a cell activity, e.g., using a marker gene, e.g., a lacZ gene or green fluorescence protein (GFP) gene, fused to the regulatory sequence of VEGF and following production of the marker.
In a preferred embodiment, the compound is a fragment or an analog of VEGF.
In a preferred embodiment, the compound is a transition metal ion.
In a preferred embodiment, VEGF transcription is evaluated in a cell, e.g., a keratinocyte, e.g., a follicular keratinocyte.
A xe2x80x9ctreatmentxe2x80x9d, as used herein, includes any therapeutic treatment, e.g., the administration of a therapeutic agent or substance, e.g., a drug.
As used herein, the term xe2x80x9csubjectxe2x80x9d refers an animal, e.g., a mammal, e.g., a human. The mammal can be a human or non-human mammal, e.g., a swine, a bird, a cat, a dog, a monkey, a goat, or a rodent, e.g., a rat or a mouse. The subject can be a transgenic animal, e.g., a transgenic rodent, e.g., a transgenic rat or mouse.
xe2x80x9cRegulatory sequencexe2x80x9d refers to any or all of the DNA sequences that controls gene expression. An example of a regulatory sequence includes: a promoter, a positive regulatory element (such as an enhancer or a DNA-binding site for a transcriptional activator); a negative regulatory element (such as a DNA-binding site for a transcriptional repressor) and an insulator.
xe2x80x9cHeterologousxe2x80x9d refers to DNA or tissue which is derived from a different species.
xe2x80x9cHeterologous regulatory sequencexe2x80x9d refers to a sequence which is not the normal regulatory sequence of that gene.
xe2x80x9cMisexpressionxe2x80x9d, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.
The terms xe2x80x9cpeptidesxe2x80x9d, xe2x80x9cproteinsxe2x80x9d, and xe2x80x9cpolypeptidesxe2x80x9d are used interchangeably herein,
The term xe2x80x9csmall moleculexe2x80x9d, as used herein, includes peptides, peptidomimetics, or non-peptidic compounds, such as organic molecules, having a molecular weight less than 2,000, preferably less than 1,000.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.